Solarbio Suksinat Dehidrogenase (SDH) Kit Uji Aktivitas

Suksinat Dehidrogenase (SDH) Kit Uji Aktivitas

Catatan: Ambil dua atau tiga sampel berbeda untuk prediksi sebelum pengujian.

Peralatan deteksi: Spektrofotometer

Kucing No: BC0950

Ukuran: 50T/48S

Komponen

Reagen I: 60 mL×1, Simpan di -20 ℃. Reagen II: 0.6 mL×1, Simpan di -20 ℃. Reagen III: 5 mL×1, Simpan di 4 ℃.

Reagen IV: Bedak×1, Simpan di 4 ℃. When the solution will be used, add it into Reagent III to dissolve for use.

Reagen V: Bedak×1, Simpan di 4 ℃. Menambahkan 4 ml air suling saat larutan akan digunakan, the unused reagents are stored at 4℃.

Reagen VI: Bedak×1, Simpan di -20 ℃. Menambahkan 3.333 ml air suling saat larutan akan digunakan, the unused reagents are stored at 4℃.

Keterangan

Suksinat Dehidrogenase (SDH, EC 1.3.5.1) banyak ditemukan pada hewan, tanaman, mikroorganisme dan sel yang dikultur. SDH is a marker enzyme of mitochondria, which is a membrane binding enzyme located in the inner membrane of mitochondria. It is also one of the key points of respiratory electron transfer and oxidative phosphorylation. In addition, it provides electrons for the respiratory chain of various prokaryotic cells.

SDH can catalyze the dehydrogenation of succinic acid to fumaric acid. The dehydrogenation can reduce 2,6-dichlorophenol indophenol (DCPIP) under the transfer of phenazine dimethyl sulfate (PMS). 2,6- DCPIP has a characteristic absorption peak at 600 nm. The reduction rate of 2,6-DCPIP is determined by the change of absorbance at 600 nm, which represents the activity of SDH enzyme.

Diperlukan tetapi tidak disediakan

Spektrofotometer, pemandian air, tabletop centrifuge, pipet yang dapat disesuaikan, mortir/homogenizer, 1 mL kuvet kaca, es dan air suling.

Protokol

SAYA. Extraction of SDH:

Accurately weigh 0.1 g of tissue or collect 5 juta sel, menambahkan 1 mL of Reagent I and 10 μl reagen II, homogenize by using homogenizer/mortar in ice bath, fully grind, sentrifugasi di 11000 ×g untuk 10 menit pada 4 ℃, Ambil supernatan dan letakkan di atas es untuk pengujian.

II. Prosedur

1. Panaskan spektrofotometer untuk 30 menit, sesuaikan panjang gelombang ke 600 nm dan atur nol dengan air suling.
2. Procedure test

Nama reagen (μL)	Tabung reaksi (T)	Tabung hitam (B)
Reagen III	60	60
Reagen V	60	60
Air sulingan	800	800
Keep warm at 25℃(general species) or 37℃(mammals) mandi air untuk 10 menit.
Sampel	30	–
Air sulingan	–	30
Reagen VI	30	30

Add each reagent to 1 mL glass cuvette in turn, and start timing at the same time of adding Reagent VI, record the initial absorbance A1 at the wavelength of 600 nm untuk 20 detik. Then put the cuvette together with the reaction solution into a water bath of 37℃ (mamalia) atau 25℃ (spesies lain), and accurate reaction for 1 menit. Quickly take out the cuvette and dry it, and record the absorbance A2 at 80 seconds at 600 nm. ΔA = A1-A2, obtain ΔAT, ΔAB.

AKU AKU AKU. Calculation of SDH activity

Calculation formula for determination with 1 mL kuvet kaca.

• Konsentrasi protein

Definisi satuan: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every milligram tissue protein.

SDH(U/mg keuntungan)＝[(ΔAT-ΔAB)−(×d)×VRV×109]−(CPR × Vs.) ÷T＝1555.556×(ΔAT-ΔAB)−Cpr

• Berat sampel

Definisi satuan: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every gram tissue.

SDH(U/g)＝[(ΔAT-ΔAB)−(×d)×VRV×109]−(Vs ÷ vst × w) ÷T＝1571.111×(ΔAT-ΔAB)−W

• Germ or cells

Definisi satuan: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every 10 thousand germ or cells.

SDH(sel U/104)＝[(ΔAT-ΔAB)−(×d)×VRV×109]−(VS÷VST×500) −T

＝3.142×(ΔAT-ΔAB)

Vrt: Volume reaksi total, 0.98×10-3 L;

e: The molar extinction coefficient of 2,6-DCPIP, 2.1×104 L/mol/cm; D: The light diameter of cuvette, 1 cm;

Vs: Volume sampel, 0.03 ml;

Vst: Add the volume of Reagent I and Reagent II, 1.01 ml; T: Waktu reaksi(menit), 1 menit;

Cpr: Konsentrasi protein sampel, mg/mL; W: Berat sampel, G;

500: Cells or germ, 5 juta.

Catatan

1. All reagents and samples shall be placed on ice during the determination to avoid denaturation and deactivation.
2. Jika ΔA lebih besar dari 0.5, the enzyme solution should be diluted with enzyme extract to obtain ΔA with less than 0.5, which can improve the detection sensitivity.
3. Because the Extract solution contains a certain concentration of protein (tentang 1 mg/mL), it is necessary to subtract the protein content of the Extract solution itself when determining the protein concentration of the sample.

Contoh eksperimental:

1. Take 0.1g of kidney, menambahkan 1 mL of Reagent Ⅰ and 10 μL Reagent Ⅱ, grind the homogenate with ice bath, centrifuge at 4℃ and 11000g for 10 menit, and place the supernatant on ice. According to the determination procedure, the enzyme activity is calculated as follows: ΔAT= A1T- A2T=0.82-0.681=0.139, ΔAB= A1B- A2B=905-0.904=0.001

SDH activity (massa U/g) = 961.905×(ΔDI- ΔAB) ÷ W =2168.13 U/g mass.

Referensi

• Fattoretti P, Bertoni-Freddari C, Caselli U, dkk. Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging[J]. Mechanisms of aging and development, 1998, 101(1-2): 175- 182.

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